Dr Piyush Tailor

Activated Partial Thromboplastin Time

( By Elite pro analyzer)

Purpose of examination:

Activated partial thromboplastin time is a general coagulation screening test of the coagulation factors XII, XI, IX, VIII, X, V, II and fibrinogen.

The activated partial thromboplastin test measures the clotting time of plasma after the activation of contact factors but without added tissue thromboplastin and so indicates the overall efficiency of the intrinsic pathway.

Principle of examination:

The APTT involves the recalcification of plasma in the presence of a standardized amount of cephalin ( platelet substitute) and a factor XII activator ( kaolin).

Principle of Instrument:

Optical Measuring System

The Loading and Analysis area also houses the optical system for analysis on

two channels: nephelometric and absorbance.

Nephelometric channel: the light source for this channel is a light emitting

diode (LED); the light (λ = 660 nm) is directed to the reaction cuvettes in the

rotor by a fiber optic system. The scattered light is read at a 90^o angle with

respect to the incident beam using a solid state detector located below the

rotor holder.

Absorbance channel: the light source is a halogen lamp, from which the

radiation is directed to the reaction cuvettes in the rotor via a quartz optic

fiber and a focusing system. The selection of the wavelength for analysis is

effected by a narrow-band interference filter centered at λ = 405 nm.

The optical detector is mounted in the cover of the loading/analysis area; therefore the readings are made at an 180^oangle from the light beam.

The optical path width for the absorbance channel is 0.5 cm (cuvette height). The absorbance values provided by the analyzer are normalized to 1 cm. These values are generally twice those ones obtained on other ACL models, for which the absorbance values are not normalized and are thus exactly the ones obtained for the 0.5 cm cuvette path.

Performance specification:

Sample type required:

Citrated plasma

Preservative needed:

Sample collected In Sodium Citrate bulb.9 volume of blood is collected in 1

volume of 3.2% trisodium citrate anticoagulant

Reagents required:

Synthasil, Hemosil

CaCl2

Cleaning solution A

Cleaning Agent B

Calibration method:

calibration is done by manufacture annually and calibration report is received in the laboratory .

Calibration reports are filed in HI: C\ Records\File\21\Calibration Records

Detailed work bench instruction:

• Switch on the main power supply.
• Switch on the ELITE Pro switch.(Back side of machine)
• Switch on the monitor.
• Monitor will display starting ACL, please wait and then system load in progress.
• For log in enter user and password.
• Database view appear on screen.
• Menu contains: ‘Analysis’ ‘QC’ ‘Calibration’ ‘Diagnostic’ ‘Setup’ ‘utility’
• Select option ‘Diagnostic’ then moves to maintenance and everyday change cleaning cycle date.
• Select option ‘Diagnostic’ then moves to cleaning and start.
• After completion of cleaning select ‘Analysis’ and move to multitest session.
• Multitest preanalysis displayed on monitor then click on circle (Number) and enter sample ID then select test like PT or APTT then confirm test.
• Daily controls are run first.
• Then for second sample just like first sample select position and enter sample ID.

• After loading all samples click on √ box then press on ‘RUN’.
• Then monitor will display ‘Pre Analysis in progress’ then pre analysis result control pannel is displayed then select ‘Continue’.
• Samples are tested automatically. Results are displayed on screen.
• After completion select cleaning and in last shut down (Screen & monitor ) and switch off (backside of machine).

Quality control procedure:

• Daily level 1 and level 2 controls run. Results of control samples are recorded in C\Records\File\9\Internal Quality Control Records
• Comparison done once a day by running a pooled normal plasma on Elite Pro coagulation analyzer and then by Manual method. Results are compared and documented.
• Inter instrument comparison done once a day by running a random sample on Elite Pro and STAGO. Results are compared and documented
• External quality assessment: Participate in external quality assessment program ( ISHTM-CMC VELLORE EQAS PROGRAM- HAEMOSTASIS ) every 4 mthly and maintain data in C\Records\File\2\Results of EQA and interlaboratory comparison

Interference:

In order to maintain the activity of various coagulation factor, the samples should be taken with care and following the professional standards in tubes with a specified concentration of citrate.

• Quality of centrifugation and storage temperature of the sample should also be carefully insured before the analysis.
• Plasma that is hemolysed, partially coagulated (presence of microclot), damaged by temperature changes or with bubbles on its surface may cause inaccurate results
• Plasma that has been frozen may contain interfering precipitates when thawed. These precipitates should be removed before measurement is performed
• Poor preparation of the reagent regarding reconstitution volume, stabilization time, stirring as well as the presence of bubbles or the accidental presence of a magnetic rod may lead to incorrect result.

Calculation of results and uncertainty:

When monitoring heparin therapy, any release of platelet factor 4 which is a potent inhibitor of heparin, represents a major source of error. In that case centrifugation should be performed within one hour of sample collection.

Biological reference interval:

27- 34 seconds

Reportable interval for examination results:

6-247 seconds

Critical values:

> one minute

Interpretation by the laboratory:

Results given by the analyzer must always be analyzed according to the patient’s history, clinical examination and to any other biological results.

The common causes of prolonged APPT are as follow:

• Disseminated intravascular coagulation
• Liver disease,
• Massive transfusion with plasma depleted red blood cells
• Administration of or contamination with heparin or other anti coagulants
• Circulating anticoagulant ( inhibitor)
• Deficiency of a coagulation factor ( factor VIII, IX,XI,XII); if all the factors are normal , a deficiency of HMW kininogen should be considered
• The APTT is also moderately prolonged in patients taking oral anticoagulant drugs and in the presence of vitamin K deficiency.
• Occasionally, a patient with previously undiagnosed haemophilia or another congenital coagulation disorder presents with an isolated prolonged APTT

Potential sources of variability:

Pre analytic variables:

• Whenever possible venous samples should be collected without a pressure cuff. Venous occlusion causes hemoconcentration and activation of some clotting factors
• Faulty collection of sample resulting in it undergoing partial clotting
• Blood samples from indwelling line or catheter should not be used for test as they are prone to dilution or heparin contamination.
• Order of draw should be taken care of while blood collection
• Underfilling or overfilling of tube or high or low hematocrit ( can cause the volume of citrate in relation to the plasma volume to be incorrect)
• Testing should be completed within 2 hours of collection; undue delay in sample analysis can cause inaccuracy

Reference:

Reference Manual of ELITE PRO

Practical Haematology (Dacie and Lewis)-twelfth edition

( By STAGO analyzer)

Purpose of examination:

Activated partial thromboplastin time is a general coagulation screening test of the coagulation factors XII, XI, IX, VIII, X, V, II and fibrinogen.

The activated partial thromboplastin test measures the clotting time of plasma after the activation of contact factors but without added tissue thromboplastin and so indicates the overall efficiency of the intrinsic pathway.

Principle of examination:

The APTT involves the recalcification of plasma in the presence of a standardized amount of cephalin ( platelet substitute) and a factor XII activator ( kaolin).

Principle of Instrument: The STA compact system performs in vitro tests for diagnosis and monitoring of pathologies linked to hemostasis.

The principle consists of measuring the variation of the ball oscillation amplitude through inductive sensors. The ball has a pendular movement obtained to the two curveted rail tracks of the cuvettes and an alternate electro-magnetic field created by two independent coils. The intensity of the magnetic field varies depending on the test to be carried out and on the expected clot.

The oscillation amplitude is constant when the environment has constant viscosity and decreases when the environment viscosity increases.

The detection system of the oscillation amplitude variation is based on two measurement coils. The transmitting coil emits an electro- magnetic field. The signal received by the receiver coil depends on the ball position in the cuvette. An algorithm uses these magnetic field variation to calculate the oscillation amplitude variation and to accurately determine the clotting time.

Performance specification:

Sample type required:

Citrated plasma

Preservative needed:

Sample collected In Sodium Citrate bulb.9 volume of blood is collected in 1

volume of 3.2% trisodium citrate anticoagulant

Reagents required:

STA DESORB U

C.K. PREST: Reagent 1: cephalin (platelet substitute) prepared from rabbit cerebral tissues

Reagent 2 : buffered suspension of kaolin

Ca Cl2

Cleaner solution

Coolant (Ethylene Glycol)

Calibration method:

calibration is done by manufacture annually and calibration report is received in the laboratory .

Calibration reports are filed in HI: C\ Records\File\21\Calibration Records

Detailed work bench instruction:

• Switch on the main power supply.
• Switch on the STAGO switch
• Switch on the monitor
• Global verification is done automatically and on completion GLOBAL VERIFICATION DONE is displayed on monitor
• To continue press ‘ENTER’
• Text box is displayed on screen: START OF TEST; Press ‘Esc’ to continue
• “Test Panel” is displayed on monitor; Press ‘Esc’ for MENU

MENU contains: status/ loading/ files/ calibration- control/ set up/ maintenance/ Halt

• Select option ‘loading’ and move on products to load the reagents. Product reagent drawer opens: reagents for a PTT ( STA DESORB U; C.K.Prest and CaCl2) are first read by barcode and then product is kept at appropriate place according to size of vial; details are entered ( reagent ID, reagent name, volume, stability, lot number and position);
• Press ‘Esc’ to return to main MENU
• Select option ‘calibration- control’; select the test option from the list
• Press ‘Esc’ for the option and select “change range” and enter the reference range and save (F10)
• Press ‘Esc’ to return to the option ; enter the access code and select option “RUN”
• Press ‘Esc’ to return to the option and select “result list”
• Press ‘Esc’ to return to main MENU and select the option “Loading” ; select samples (F1); sample drawer will open. Samples are loaded by giving ID and sample position; Test is selected and validated by pressing F10
• Samples are tested automatically. When the results are displayed on screen printout taken

Quality control procedure:

• Control plasma (Freeze dried pooled plasma of 20 normal individual) is run in duplicate as normal control with every batch and mean is derive and recorded.
• Inter instrument comparison done once a day by running a random sample on Elite Pro and STAGO. Results are compared and documented.

Interference:

In order to maintain the activity of various coagulation factor, the samples should be taken with care and following the professional standards in tubes with a specified concentration of citrate.

• Quality of centrifugation and storage temperature of the sample should also be carefully insured before the analysis.
• Plasma that is hemolysed, partially coagulated (presence of microclot), damaged by temperature changes or with bubbles on its surface may cause inaccurate results
• Plasma that has been frozen may contain interfering precipitates when thawed. These precipitates should be removed before measurement is performed
• Poor preparation of the reagent regarding reconstitution volume, stabilization time, stirring as well as the presence of bubbles or the accidental presence of a magnetic rod may lead to incorrect result.

Calculation of results and uncertainty:

When monitoring heparin therapy, any release of platelet factor 4 which is a potent inhibitor of heparin, represents a major source of error. In that case centrifugation should be performed within one hour of sample collection.

Biological reference interval:

27- 34 seconds

Reportable interval for examination results:

20-180 seconds

Critical values:

> one minute

Interpretation by the laboratory:

Results given by the analyzer must always be analyzed according to the patient’s history, clinical examination and to any other biological results.

The common causes of prolonged APPT are as follow:

• Disseminated intravascular coagulation
• Liver disease,
• Massive transfusion with plasma depleted red blood cells
• Administration of or contamination with heparin or other anti coagulants
• Circulating anticoagulant ( inhibitor)
• Deficiency of a coagulation factor ( factor VIII, IX,XI,XII); if all the factors are normal , a deficiency of HMW kininogen should be considered
• The APTT is also moderately prolonged in patients taking oral anticoagulant drugs and in the presence of vitamin K deficiency.
• Occasionally, a patient with previously undiagnosed haemophilia or another congenital coagulation disorder presents with an isolated prolonged APTT

Potential sources of variability:

Pre analytic variables:

• Whenever possible venous samples should be collected without a pressure cuff. Venous occlusion causes hemoconcentration and activation of some clotting factors
• Faulty collection of sample resulting in it undergoing partial clotting
• Blood samples from indwelling line or catheter should not be used for test as they are prone to dilution or heparin contamination.
• Order of draw should be taken care of while blood collection
• Underfilling or overfilling of tube or high or low hematocrit ( can cause the volume of citrate in relation to the plasma volume to be incorrect)
• Testing should be completed within 2 hours of collection; undue delay in sample analysis can cause inaccuracy

Reference:

Reference Manual of STA compact (STAGO)

Practical Haematology (Dacie and Lewis)-tenth edition

Reagent insert of STAGO reagent( C.K. PREST)

(Manual Method)

Purpose of examination:

Activated partial thromboplastin time is a general coagulation screening test of the coagulation factors XII, XI, IX, VIII, X, V, II and fibrinogen.

The activated partial thromboplastin test measures the clotting time of plasma after the activation of contact factors but without added tissue thromboplastin and so indicates the overall efficiency of the intrinsic pathway.

Principle of examination:

The APTT involves the recalcification of plasma in the presence of a standardized amount of cephalin ( platelet substitute) and a factor XII activator ( kaolin).

Principle of method:

Cephaloplastin activates the coagulation factors of the intrinsic pathway of the coagulation mechanism in the presence of calcium ions. APTT is prolonged by deficiency of one or more of the clotting factors of the intrinsic pathway and in the presence of coagulation inhibitors like heparin.

Performance specification:

Sample type required:

Citrated plasma(9 parts of blood & 1 part of citrate)

Preservative needed:

Sample collected In Sodium Citrate bulb.9 volume of blood is collected in 1volume of 3.2% trisodium citrate anticoagulant

Reagents:

1. CELIN-SE (a liquid ready to use activated cephaloplastin reagent)

2. Calcium chloride solution

Equipments :

Water bath, Thermometer, Bulb, Stopwatch, Pipette (100 ul & 200 ul), test cuvette

Detailed work bench instructions:

1. Bring the required reagent to the room temperature.
2. Prewarm required volume of reagents (CELIN-SE & CaCl2) for immediate use in clean & dry test tube at 37⁰ C
3. Pipette 100 μl of test plasma & control into test cuvette at 37⁰ C.
4. Pipette 100μl of the prewarmed CELIN-SE into test & control cuvette.
5. Mix well & incubate at 37⁰C for 10 min.
6. Forcibly pipette 100μl of prewarmed CaCl2 into test & control cuvette. Start stop watch simultaneously. Shake the tube briefly to mix the content, keep at 37 degree C for 20 seconds.
7. Following 20 seconds incubation, remove the tube gently tilt back and forth until gel clot forms, stop the watch & record the clotting time in seconds.

Quality control procedure:

• Control plasma (Freeze dried pooled plasma of 20 normal individual) is run in duplicate as normal control with every batch and mean is derive and recorded.
• Comparison done once a day by running a pooled normal plasma on Elite Pro coagulation analyzer and then by Manual method. Results are compared and documented

Interference:

In order to maintain the activity of various coagulation factor, the samples should be taken with care and following the professional standards in tubes with a specified concentration of citrate.

• Quality of centrifugation and storage temperature of the sample should also be carefully insured before the analysis.
• Plasma that is hemolysed, partially coagulated (presence of microclot), damaged by temperature changes or with bubbles on its surface may cause inaccurate results
• Plasma that has been frozen may contain interfering precipitates when thawed. These precipitates should be removed before measurement is performed
• Poor preparation of the reagent regarding reconstitution volume, stabilization time, stirring as well as the presence of bubbles or the accidental presence of a magnetic rod may lead to incorrect result.

Calculation of results and uncertainty:

When monitoring heparin therapy, any release of platelet factor 4 which is a potent inhibitor of heparin, represents a major source of error. In that case centrifugation should be performed within one hour of sample collection.

Biological reference interval:

27- 34 seconds

Reportable interval for examination results:

1-180 seconds

Critical values:

> one minute

Interpretation by the laboratory:

Results given by the analyzer must always be analyzed according to the patient’s history, clinical examination and to any other biological results.

The common causes of prolonged APPT are as follow:

• Disseminated intravascular coagulation
• Liver disease,
• Massive transfusion with plasma depleted red blood cells
• Administration of or contamination with heparin or other anti coagulants
• Circulating anticoagulant ( inhibitor)
• Deficiency of a coagulation factor ( factor VIII, IX,XI,XII); if all the factors are normal , a deficiency of HMW kininogen should be considered
• The APTT is also moderately prolonged in patients taking oral anticoagulant drugs and in the presence of vitamin K deficiency.
• Occasionally, a patient with previously undiagnosed haemophilia or another congenital coagulation disorder presents with an isolated prolonged APTT

Potential sources of variability:

Pre analytic variables:

• Whenever possible venous samples should be collected without a pressure cuff. Venous occlusion causes hemoconcentration and activation of some clotting factors
• Faulty collection of sample resulting in it undergoing partial clotting
• Blood samples from indwelling line or catheter should not be used for test as they are prone to dilution or heparin contamination.
• Order of draw should be taken care of while blood collection
• Underfilling or overfilling of tube or high or low hematocrit ( can cause the volume of citrate in relation to the plasma volume to be incorrect)
• Testing should be completed within 2 hours of collection; undue delay in sample analysis can cause inaccuracy

REFERENCE :

Practical Haematology (Dacie and Lewis)-Twelfth edition (page no.382)

Literature from CELIN – SE kit